Evaluation of bactericidal and fungicidal activity of ferrocenyl or phenyl derivatives...

concentration at which there was no visible growth (turbidity) was
taken as the MIC. Then from each tube, one loopful was cultured on
Plate count agar and incubated for 24 h at 30 C. The lowest
concentration of the compound supporting no colony formation
was defined as the MBC. Doxycycline was used as standard antibiotic.
Solutions of the test compounds and doxycycline were
prepared in ethanol at concentration of 2 mg mL 1 . The two fold
dilutions of the compounds were prepared (200, 100, 50, 25,
12.5 mg mL 1 ). The microorganism suspensions at 10 5 CFU/mL
concentrations were inoculated into the corresponding test tube.
4.13. Measurement of octanol/water partition coefficient (log Po/w)
The log Po/w values of the compounds were determined by
reverse-phase HPLC on a C-8 column (Kromasil C8, from Macherey
Nagel, France) according to the method previously described by
Minick [55] and Pomper log Po/w. Measurement of the chromatographic
capacity factors (k0) for each compounds were done at
various concentrations in the range 95e70% methanol (containing
0.25% octanol) and an aqueous phase consisting of 0.15% n-decylamine
in 0.02 M MOPS (3-morpholinopropanesulfonic acid) buffer
pH 7.4 (prepared in 1-octanol-saturated water). These capacity
factors (k 0 ) are extrapolated to 100% of the aqueous component
given the value of k 0 w. log Po/w (y) is then obtained by the formula:
log log Po/w ¼ 0.13418 þ 0.98452 log k 0 w.
4.14. Antiproliferative tests on hormone-independent breast cancer
cells MDA-MB-231
Stock solutions (1 10 2 M) and serial dilutions of the
compounds to be tested were prepared in DMSO just prior to use.
Dulbecco’s modified eagle medium (DMEM), fetal calf serum,
glutamine and kanamycine were obtained from Invitrogen. MDA-
MB-231 cells were from ATCC LGC standards. Cells were maintained
in a monolayer culture in DMEM with phenol red/Glutamax I supplemented
with 9% fetal bovine serum at 37 C in a 5% CO2/airhumidified
incubator. For proliferation assays, MDA-MB-231 cells
were plated in 1 mL of DMEM without phenol red, supplemented
with 9% decomplemented and hormone-depleted fetal bovine
serum, 0.9% kanamycin, 0.9% Glutamax I and incubated. The
following day (D0), 1 mL of the same medium containing the
compounds to be tested was added to the plates. After 3 days (D3)
the incubation medium was removed and 2 mL of the fresh medium
containing the compounds was added. After 5 days the total protein
content of the plate was analyzed as follows: cell monolayers were
fixed for 1 h at room temperature with methylene blue (1 mg mL 1 in
50:50 water/MeOH mixture), then washed with water. After addition
of HCl (0.1 M, 2 mL), the plate was incubated for 1 h at 37 C and
then the absorbance of each well (three wells for each concentration)
was measured at 655 nm with a Biorad microplate reader. The
results are expressed as the percentage of proteins vs. the control.
Experiments were performed at least in duplicate.
Acknowledgements
We thank the Ministère de la Recherche and the Centre National
de la Recherche Scientifique for financial support, Mrs Barbara
McGlinchey for helpful discussions. M. El Arbi thanks the Ministère
de l’Enseignement Supérieur et de la Recherche Scientifique of
Tunisia for financial support and ISET-Sfax for technical support.
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